In an integrated program of laboratory and clinical investigation, we study the molecular biology of the heritable connective tissue disorders osteogenesis imperfecta (OI). Our objective is to elucidate the mechanisms by which the primary gene defect causes skeletal fragility and then apply the knowledge gained from our studies to the treatment of children with these conditions. Structural defects of type I collagen molecule are well known to cause the dominant bone disorder OI. Ten years ago we identified defects in two components of the collagen prolyl 3-hydroxylation complex, CRTAP and P3H1 (encoded by LEPRE1) as the cause of recessive OI. Our work generated a new paradigm for collagen-related disorders of matrix, in which structural defects in collagen cause dominant OI, while defects in proteins that interact with collagen cause the rare forms of OI. Recessive OI is now a major area of investigation for the BEMB. The phenotypes of types VII and VIII OI are distinct from classical dominant OI, but difficult to distinguish from each other. We showed that mutual stabilization of CRTAP and P3H1 underlies the phenotypic and biochemical similarity of types VII and VIII OI. With collaborators at the Boltzman Osteology Institute, we recently focused on the bone of the non-lethal subset of type VIII OI patients. Bone histology was similar to type VII OI, although it had the distinctive feature of extremely thin trabeculae and patches of increased osteoid, suggesting mineralization is slower in type VIII than VII OI. BMDD yielded increased mineralization of type VIII bone, as in classical OI and type VIII OI, but the proportion of bone with low mineralization was increased in type VIII bone vs type VII. Type IX OI has a distinctive phenotype without rhizomelia, and distinctive biochemistry. We generated a CyPB KO mouse, which has reduced bone density and strength, but increased brittleness. Collagen folds more slowly in the absence of CyPB, but CsA treatment revels the potential existence of another collagen PPIase. CyPB supports LH1 activity and in its absence there is significant reduction of hydroxylation of crosslinking residue K87, which alters fibril structure and reduces bone strength. With collaborators at the University of North Carolina we showed that CyPB interacted with all LH forms (LH1-3). In tendon, CyPB modulates collagen cross-linking by differentially affecting lysine hydrozylation in the helical and telopeptide domains of collagen. While lysine hydroxylation was decreased in the collagen helix, it was increased in the telopeptide cross-link sites. CyPB modulates crosslinking by affecting lysine hydroxylation in a site-specific manner. We have extended our work on PPIB function in a collaboration with investigators in Jerusalem. CyPB was shown to be critical to folding presenilin-1, a protein linked to familial Alzheimers Disease. Some substitutions in presenilin-1 make it resistant to folding properly by CyPB. Conversely, the brains of CyPB knock-out mice were shown to have reduced quantities of processed, active presenilin-1. These Prion protein-containing aggresomes are cytosolic components of an ER quality control mechanism in which the Golgi plays no part. We delineated a mutation in IFITM5, which encodes the transmembrane protein BRIL, that establishes a connection between types V and VI OI. The BRIL S40L substitution results in minimal expression and secretion of PEDF by mutant FB and osteoblasts. Om contrast to the gain-of-function BRIL mutation that causes type V OI, the BRIL S40 causes decreased mineralization and expression of bone markers. Only type I collagen shows similar expression pattern in both mutations, with decreased expression, secretion and matrix incorporation. Type XIV OI is a moderately severe form of OI which was identified in 2013. It is caused by recessive defects in TMEM38B, which encodes TRIC-B, an ER cation channel. We identified probands with recessive null defects in TRIC-B and studied the function of TRIC-B deficiency in their fibroblasts and osteoblasts. TRIC-B deficiency impaired ER calcium flux which resulted in ER-stress along the ATF4 pathway. TRIC-deficiency was demonstrated to be collagen related because it impaired collagen synthesis and assembly at multiple steps. Collagen helical lysine hydroxylation was reduced, although the levels of LH1 protein were increased.. We also investigated the unique aspects of TMEM38B deficiency phenotype in 8 patients with type XIV OI in collaboration with colleagues in the UK and Vienna. There is striking variability of OI severity even between siblings. Multiple patients demonstrated various cardiac defects suggesting that TMEM38B might supplement TMEM38A in cardiac pathology. Like other OI types, type XIV patients have low DXA BMD. However, unusual among OI types, bone matrix mineralization is not increased and nanopososity is low. Using cells cultured from proband bone, we demonstrated a complex osteoblast differentiation defect with decreased expression of early markers and increased expression of late and mineralization related markers. Type XIV osteoclasts likely have an intrinsic defect because of expression of TMEMB; this would explain low bone turnover in type XIV OI. This year we delineated the first X-linked recessive form of OI, made even more exciting by its novel bone mechanism. X-linked OI is moderately severe with pre- and post-natal fractures of ribs and long bones, dysplastic bone with bowing and crumpling. It is caused by missense mutations in MBTPS2, which encodes Site 2 protease (S2P). S2P is a critical component of Regulated Intramembrane Proteolysis (RIP), a process in which S1P and S2P located in the Golgi Membrane sequentially cleave regulatory proteins that are transported from the ER membrane in times of cell stress or sterol metabolite deficiency. The S2P substitutions in X-OI are located in or near the S2P motif critical for metal ion coordination. The levels of S2P transcripts and protein are normal but RIP function on substrates OASIS, ATF6 and SREBP are impaired. At the bone tissue level, hydroxylation of collagen K87 residues in type I collagen is reduced by half, altering collagen crosslinking in bone. The osteoblasts with S2P defects also have a differentiation defect.